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Published in final edited form as: Ann Emerg Med. 2010 April ; 55(4): 352–363. doi:10.1016/j.annemergmed.2009.12.002.
Intramuscular Cobinamide Sulfite in a Rabbit Model of Sub-Lethal Cyanide Toxicity Matthew Brenner, MD1,2, Jae G. Kim, PhD1, Sari B. Mahon, PhD1,2, Jangwoen Lee, PhD1, Kelly A. Kreuter, MS1, William Blackledge, MD3, David Mukai, BS1, Steve Patterson, PhD4, Othman Mohammad, MD3, Vijay S. Sharma, PhD3, and Gerry R. Boss, MD3 1 Laser Microbeam and Medical Program, Beckman Laser Institute and Medical Clinic, University of California, Irvine, California 92612-1475 2 Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California,
Irvine, California 92868
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3
Department of Medicine, University of California, San Diego, La Jolla, CA, 92093-0652
4
Department of Pharmacology, University of Minnesota, Minneapolis, MN
Abstract Objective—To determine the ability of an intramuscular cobinamide sulfite injection to rapidly reverse the physiologic effects of cyanide toxicity. Background—Exposure to cyanide in fires and industrial exposures and intentional cyanide poisoning by terrorists leading to mass casualties is an ongoing threat. Current treatments for cyanide poisoning must be administered intravenously, and no rapid treatment methods are available for mass casualty cyanide exposures. Cobinamide is a cobalamin (vitamin B12) analog with an extraordinarily high affinity for cyanide that is more water-soluble than cobalamin. We investigated the use of intramuscular cobinamide sulfite to reverse cyanide toxicity induced physiologic changes in a sublethal cyanide exposure animal model.
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Methods—New Zealand white rabbits were given 10 mg sodium cyanide intravenously over 60 minutes. Quantitative diffuse optical spectroscopy and continuous wave near infrared spectroscopy monitoring of tissue oxy- and deoxyhemoglobin concentrations were performed concurrently with blood cyanide level measurements and cobinamide levels. Immediately after completion of the cyanide infusion, the rabbits were injected intramuscularly with cobinamide sulfite (n=6) or inactive vehicle (controls, n=5). Results—Intramuscular administration led to rapid mobilization of cobinamide and was extremely effective at reversing the physiologic effects of cyanide on oxyhemoglobin and deoxyhemoglobin extraction. Recovery time to 63% of their baseline values in the central nervous system was in a mean of 1032 minutes in the control group and 9 minutes in the cobinamide group with a difference of 1023 minutes (95% confidence interval [CI] 116, 1874 minutes). In muscle tissue, recovery times were 76 and 24 minutes with a difference of 52 minutes (95% CI 7, 98min). Red blood cell cyanide
© 2009 Published by Mosby, Inc on behalf of American College of Emergency Physicians. Address for reprints: Dr. Matthew Brenner,
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levels returned towards normal significantly faster in cobinamide sulfite-treated animals than in control animals.
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Conclusions—Intramuscular cobinamide sulfite rapidly and effectively reverses the physiologic effects of cyanide poisoning, suggesting that a compact cyanide antidote kit can be developed for mass casualty cyanide exposures.
Introduction The development of cyanide toxicity may occur from smoke inhalation, industrial exposure, and acts of terrorism. (1-4). Doses of as little as 50 mg may be fatal to humans, with more than 5.2 billion pounds of cyanide produced annually worldwide (5). Lethal exposures can occur from cyanide ingestion or inhalation, and irreversible injury or death can occur within minutes of exposure. Mass casualty cyanide exposure from intentional terrorism acts is a major concern to civilian and military personnel. Terrorist plans to attack passengers in the NY subway system using cyanide were discovered by the United States intelligence authorities in 2003 (6).
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Current treatments for cyanide poisoning include three general classes of agents: methemoglobin generators (sodium nitrite, amyl nitrite, and dimethyl aminophenol), sulfur donors (sodium thiosulfate and glutathione), and direct binding agents (hydroxocobalamin and dicobalt edetate) (4,7). These drugs are effective for cyanide exposure of only a small number of victims concurrently, because they must be administered intravenously by skilled personnel. Given the need for immediate treatment of cyanide exposed-persons, and the ever present danger of mass casualties from cyanide poisoning, rapidly acting cyanide antidotes that can be administered simply are desperately needed. An ideal candidate for treating cyanide poisoning would have a long shelf-life, exhibit minimal toxicity, i.e., have a high therapeutic index, and could be administered by minimally trained individuals or by self-administration, e.g., intramuscular injection.
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Cobinamide is a potential agent for treating cyanide poisoning (8) that may satisfy these criteria. It is the penultimate precursor in cobalamin biosynthesis, lacking the dimethyl-benzimidazole ribonucleotide tail coordinated to the cobalt atom in the lower axial position (Figure 1). Thus, whereas cobalamin has only an upper ligand binding site, cobinamide has both an upper and lower ligand binding site. Moreover, the dimethylbenzimidazole group has a negative transeffect on the upper binding site, thereby reducing cobalamin's affinity for ligands (9). The combined effect is that each cobinamide molecule can bind two cyanide molecules, and that cobinamide has a much greater affinity for cyanide than cobalamin, with a KA overall of ≈1022 M-1 [binding affinity for first cyanide ion is 1014 M-1 and for second ion is 108 M-1] (10). This suggests that cobinamide should be a more effective cyanide detoxifying agent than cobalamin, as we found in previous work (8). In aqueous solution, cobinamide exists as aquohydroxocobinamide, which we show is at least five times more water soluble than cobalamin. The combination of cobinamide's high binding affinity for cyanide, binding of two cyanide molecules, and relatively high water solubility suggest it could be administered in concentrated enough solutions for intramuscular injection. In addition, cobinamide is stable at room temperature, and we provide evidence that cobinamide sulfite exhibits minimal toxicity to mice and rats at doses as high as 300-800 mg/kg. For these reasons, we assessed the feasibility of intramuscular injections of cobinamide sulfite in a cyanide toxic animal model (11). In addition to developing cyanide countermeasures, new methods to rapidly recognize and monitor cyanide exposure are essential to optimize treatment and determine therapeutic efficacy (12,13). Optical technologies such as diffuse optical spectroscopy (DOS) and continuous wave near infrared spectroscopy (CWNIRS) may provide these capabilities. The cyanide anion binds to the iron in cytochrome oxidase, blocking electron transport and, thereby, interrupting cellular respiration (4,7,14-17). Because cytochrome oxidase accounts for > 90% Ann Emerg Med. Author manuscript; available in PMC 2011 April 1.
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of cellular oxygen consumption, oxygen is no longer consumed by cyanide-poisoned tissues, and the oxygen content increases in both arterial and venous blood (18). DOS can simultaneously measure tissue optical scattering and absorption, providing accurate quantitative measures of tissue oxy- and deoxyhemoglobin (11). We have used DOS to noninvasively measure the physiologic effects of cyanide poisoning in a non-lethal rabbit model of cyanide poisoning(11). We hypothesized that intramuscular injection of cobinamide will rapidly reverse the physiologic effects of cyanide toxicity in the rabbit model and suggest that cobinamide sulfite may be a promising candidate agent for treating mass casualties from cyanide poisoning.
Methods General preparation Pathogen-free New Zealand White rabbits weighing 3.5-4.5 kg (Western Oregon Rabbit Supply, Philomath, Oregon), were used, and all procedures were reviewed and approved by the University of California, Irvine, Institutional Animal Care and Use Committee (IACUC). The methods for cyanide induction and DOS monitoring have been described previously and are summarized here (11).
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Animals were anesthetized with an intramuscular injection of a 2:1 ratio of ketamine HCl (100 mg/ml, Ketaject, Phoenix Pharmaceutical Inc., St. Joseph, MI): xylazine (20 mg/ml, Anased, Lloyd Laboratories, Shenandoah, IA) at a dose of 0.75 cc/kg using a 23 gauge 5/8 inch needle. After the intramuscular injection, a 23 gauge 1 inch catheter was placed in the animals’ marginal ear vein to administer continuous IV anesthesia. Torbutrol, 0.1-0.5mg/kg , was given subcutaneously, and then the animals were intubated with a 3.0 cuffed endotracheal tube secured by a gauze tie; they were mechanically ventilated (dual phase control respirator, model 32A4BEPM-5R, Harvard Apparatus, Chicago, IL) at a respiration rate of 32/minutes, a tidal volume of 50 cc, and FiO2 of 100%. A pulse oximeter (Biox 3700 Pulse Oximeter, Ohmeda, Boulder, CO) with a probe was placed on the tongue to measure SpO2 and heart rate. Blunt dissection was performed to isolate the femoral artery and vein on the left thigh for blood sampling, cyanide infusion, and systemic pressure monitoring. Sodium cyanide (10 mg) dissolved in 60 ml of phosphate-buffered saline was given intravenously over 60 minutes; this dose of cyanide (~2.5 mg/kg) is non-lethal to rabbits, but does cause marked changes in tissue oxy- and deoxyhemoglobin concentrations (11). On completion of the experiment, the animals were euthanized with an intravenous injection of 1 ml Euthasol (1.0cc, Euthasol, Virbac AH, Inc. Fort Worth, Texas. 390 mg pentobarbital sodium, 50 mg phenytoin sodium/ml) administered through the marginal ear vein.
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Venous blood cyanide levels were measured at baseline, 5 minutes prior to completion and at the end of the cyanide infusion, and at 2.5, 5, 7.5, 10, 15, 30, 45, and 60 minutes after injecting cobinamide sulfite. During this time, DOS and CWNIR measurements were taken continuously, with each measurement set requiring an average of 30 sec to complete. Study and control groups A total of 11 animals were studied: five animals (control group) received either no intramuscular injection (four animals) or an intramuscular injection of 12.5 mg sodium sulfite dissolved in 1 ml phosphate-buffered saline (one animal), and six animals received an intramuscular injection of 83.5 mg cobinamide (0.082 millimole) mixed with 12.5 mg sodium sulfite in 1 ml phosphate-buffered saline immediately following completion of the cyanide infusion and DOS measurements. The cobinamide dose was calculated to achieve a molar
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equivalent for cyanide neutralization, and the injections were performed intramuscularly in the right gluteus or left pectoralis muscle.
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Non-invasive measurements using diffuse optical spectroscopy (DOS)
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Diffuse optical spectroscopy (DOS) measurements were obtained through a fiberoptic probe with a light diode emitter and detector at a fixed distance (10 mm) from the source fiber, which was placed on the shaved surface of the right inner thigh of the animal. The broadband DOS system we constructed (11,19-23) combines multi-frequency domain photon migration with time-independent near infrared spectroscopy to accurately measure bulk tissue absorption and scattering spectra. It employs six laser diodes at discrete wavelengths (661,681,783, 805, 823, and 850 nm), and a fiber coupled avalanche photo diode (APD) detector (Hamamatsu highspeed APD module C5658, Bridgewater, NJ) for the frequency domain measurements. The APD detects the intensity-modulated diffuse reflectance signal at modulation frequencies from 50 to 550 MHz after propagation through the tissue. Absorption and reduced scattering coefficients are measured directly at each of the six laser diode wavelengths using frequencydependent phase and amplitude data. Reduced scattering coefficients are calculated as a function of wavelength throughout the near infrared region by fitting a power-law to six reduced scattering coefficients. Steady-state acquisition was accomplished using a broadband reflectance measurement from 650 to 1000 nm that follows frequency domain measurements using a tungsten-halogen light source (Ocean Optics HL-2000, Dunedin, FL) and a spectrometer (BWTEK BTC611E, Newark, DE). Intensity of the steady-state reflectance measurements are calibrated to the frequency domain values of absorption and scattering to establish the absolute reflectance intensity (11,19,20). Tissue concentrations of oxy- and deoxyhemoglobin are calculated by a linear least squares fit of the wavelength-dependent extinction coefficient spectra of each chromophore. We used oxy- and deoxyhemoglobin absorption spectra reported by Zijlstra et al (24) for subsequent fitting and analysis. Continuous wave near infrared spectroscopy (CWNIRS) CWNIRS was used to assess oxy- and deoxyhemoglobin effects of cyanide toxicity and reversal in the brain region. Continuous wave near infrared spectroscopy (CWNIRS) provides rapid, real time measures of tissue oxy- and deoxyhemoglobin concentration changes and penetrates more deeply into tissues than DOS (25); it can , therefore, be used to assess regions such as the CNS, an area particularly sensitive to cyanide toxicity. CWNIRS, however, does not account for scattering effects, and provides only relative information on changes in the concentrations of molecular species (as opposed to absolute concentrations obtained by DOS).
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The CWNIRS system consists of a light source (Ocean Optics HL 2000HP, Dunedin, FL), a CCD spectrometer (BWTEK BTC111E, Newark, DE), and customized optical fiber guides (1mm diameter with 10 feet length, RoMack Inc, VA) (25). Continuous wave near infrared light was delivered to the rabbit brain/CNS using a fiber optic probe placed over the forehead, and transmitted light intensities at five wavelengths (732, 758, 805, 840, 880 nm) were measured using the CCD spectrometer every second. We quantified changes in oxy- and deoxyhemoglobin concentrations throughout the experiment using Labview real time display software, and a modified Beer-Lamberts’ law (Labview 7.0, National Instrument, TX). Since it does not account for scattering effect, the unit of oxy- and deoxyhemoglobin concentration is mM/DPF where DPF is a differential pathlength factor (26). Calculated changes in oxyand deoxyhemoglobin were displayed on a computer in real time (25). Measurement of Red Blood Cell (RBC) Cyanide Concentration Cyanide in blood is bound almost exclusively to ferric(met)hemoglobin in RBCs; thus, blood cyanide can be measured by separating RBCs from plasma, and acidifying the RBCs to release cyanide as HCN gas (27). Immediately after drawing blood from the rabbits, samples were Ann Emerg Med. Author manuscript; available in PMC 2011 April 1.
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cooled to 4 °C, centrifuged, and the plasma and RBC fractions separated. All samples were kept at 4 °C and analyzed within 48 hours. For analysis, the RBCs were lysed in ice-cold water. The lysates were placed into glass tubes sealed with stoppers holding plastic center wells (Kontes Glass Co., Vineland, NJ) containing 0.1 M sodium hydroxide (NaOH). A volume of 10% trichloroacetic acid equal to the lysate was injected through the stopper into the tubes, and the tubes were shaken at 37 °C for 75 minutes. After cooling to room temperature, cyanide trapped in the NaOH was measured in a spectrophotometric assay following its reaction with p-nitrobenzaldehyde and o-dinitrobenzene at 560 nm (28). Concentrations were determined from standard curves using freshly prepared KCN dissolved in 0.1 M NaOH. Duplicate samples showed < 15% variation. Measurement of Plasma Cobinamide Concentration
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Plasma cobinamide concentrations were determined by scanning diluted plasma samples between 300 and 600 nm on an Uvikon 960 spectrophotometer (NorthStar Scientific, Leeds, England, NorthStar Scientific Limited - www.nstaruk.com). Cobinamide and dicyanocobinamide have distinctive spectra in this range. To convert all cobinamide in the plasma to one species, i.e., dicyanocobinamide, excess KCN was added to the samples. The cobinamide concentration was calculated based on a least squares regression linear-fit to standard curves generated by adding dicyanocobinamide of known concentrations to baseline plasma samples. Samples were diluted appropriately to assure they were in the linear range of the standard curve, with duplicate measurements performed on each sample. Cobinamide Solubility and Toxicity We found that cobinamide sulfite is soluble to at least 350 mM in water, whereas hydroxocobalamin was soluble to about 70 mM. These experiments were performed by dissolving known amounts of the two compounds in the minimal amount of water necessary to achieve a clear homogenous solution. The calculated concentrations were checked spectrophotometrically at 348.5 and 352 nm for cobinamide sulfite and hydroxocobalamin, respectively, using an extinction coefficient of 2.8 × 104. Cobinamide sulfite could be administered at doses of 300 and 800 mg/kg to rats and mice, respectively, without evidence of significant toxicity. These doses are about 15 and 40 times higher, respectively, than the doses used in the current study. Formal toxicology studies are currently underway. Statistical Methods
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With a two-tailed alpha of 0.05 and 5 and 6 animals in control and cobinamide sulfite group, respectively, we calculated an estimated power of 0.8 to detect a 50% difference in recovery times between groups. Baseline parameters across groups were compared using analysis of variance. Response to treatment across the groups was compared using analysis of variance with repeated measures for cyanide level comparisons. A confidence interval [CI] of 95% was considered significant. Time constants for DOS or CWNIRS changes in tissue hemoglobin oxygenation parameters were compared using analysis of variance. All data were analyzed using a standard statistical package (Systat-12, Systat Software, Inc., Chicago, IL 60606).
Results DOS monitoring of peripheral muscle region cyanide toxicity and recovery The deoxygenated hemoglobin concentration in peripheral muscle fell progressively during the 60 minutes of cyanide infusion [Fig. 2a; fractional changes in deoxyhemoglobin (deoxyhemoglobin) concentration from the baseline (pre cyanide infusion) concentration are
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shown for a representative animal]. A fall in deoxygenated hemoglobin would be expected, because of cyanide inhibiting cellular respiration and decreasing tissue oxygen consumption. When the cyanide infusion was stopped at 60 minutes, the deoxyhemoglobin concentration slowly returned over >60 minutes towards baseline values in control non-cobinamide sulfitetreated animals (Fig. 2a, grey line). The rate of return of deoxyhemoglobin levels in the cobinamide sulfite-treated animals was much faster than in control animals, particularly in the first 10 minutes after the intramuscular injection, which would be a critical time period for recovery (Fig. 2a, black line).
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Changes in deoxyhemoglobin concentrations for all animals during the recovery/treatment phase are shown in Fig. 2b, which illustrates the progression of fractional change in deoxyhemoglobin during the 60 minutes following injection of phosphate buffered saline (control) or cobinamide sulfite. Post cyanide infusion fractional deoxyhemoglobin values of the control was in a mean of -23.7%, compared to -28.7% in treatment animals; a difference of 5% (95% confidence interval [CI] -15.7, 4.6%), where the deoxyhemoglobin concentration at the completion of the cyanide infusion and start of the treatment period is designated as zero. From the data in Fig. 2b, the time constant for recovery of deoxygenated hemoglobin to baseline, pre-cyanide poisoning levels is calculated as: (~ (1-exp(-t/τ), where τ is a time constant). τ from fittings are listed below in Table 1 to determine the effective “time constant” to recovery where W is the classic time to recovery of an exponential decay fit curve to 63.2% of baseline (25). Using this formula, the time constant for recovery of deoxygenated hemoglobin was in a mean of 76 minutes in control animals, compared to 24 minutes for cobinamide sulfite-treated animals; a difference of 52 minutes (95% CI -5, 110 minutes). In the crucial first 10 minutes following cyanide poisoning, the slope of recovery of deoxygenated hemoglobin for the cobinamide sulfite-treated group is almost three times greater than for controls (Table 1). CWNIRS monitoring of CNS region cyanide toxicity and recovery
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CWNIRS monitoring over the CNS region yielded analogous results to DOS monitoring over muscle, but with a more pronounced effect of cyanide and response to treatment. Specifically, cobinamide sulfite markedly increased the rate of recovery of CNS region deoxyhemoglobin toward baseline values (Fig. 3a). The time constant for recovery of deoxyhemoglobin concentrations was a mean > 1000 minutes in control animals and 9 minutes in cobinamide sulfite-treated animals with a difference of > 900 minutes (95% CI 116, 1874 minutes) (Table 1). In the critical first 10 minutes after the end of the cyanide infusion when cobinamide sulfite was injected, the slope of recovery of the deoxyhemoglobin concentration was almost four times greater in the cobinamide sulfite-treated animals than in controls (Table 1). CNS region oxyhemoglobin returned to baseline much faster in the cobinamide sulfite-injected animals than in control animals (Fig. 3b). The time constant for returning oxyhemoglobin concentrations was in a mean > 250 minutes in control animals and 10 minutes for cobinamide sulfite-treated animals with a difference of 250 minutes (95% CI 28, 358 minutes) (Table 1). Again, the rate of return of the oxyhemoglobin concentration during the first 10 minutes after stopping the cyanide infusion in the CNS region was about four times greater in the cobinamide sulfite-treated animals than in control animals (Table 1). Total Tissue Hemoglobin Levels Total tissue hemoglobin levels measured by DOS did not change during the post cobinamide sulfite injection recovery time or in control animals during the recovery period. The changes in total hemoglobin concentration during the post cobinamide sulf ite injection recovery time or in control animals are -0.6 μM (95% CI -2.2, 1.1μM) and -1.1 μM (95% CI -2.4, 0.2 μM), respectively.
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Red blood cell cyanide concentrations
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The concentration of cyanide in red blood cells decreased significantly more rapidly following intramuscular cobinamide sulfite injection than in control animals (Fig. 4). At 30 minutes, cyanide concentrations had fallen to in a mean 68 % of peak values in cobinamide sulfitetreated animals compared to 82 % in control animals with a difference of 14 % (95% CI -35, 6 %). Analysis of variance with repeated measures revealed a significant difference between the cobinamide sulfite-treated group and controls over time (F=5.7, p
